Abstract

In the preparation process of Chinese tonic wines, the amounts of active substances released from medicinal materials decide the quality. Taking Ma-Zu-Wan-Show-Yao-Jyo as the target of the study, our aims are to investigate the effects of the released substances under the influences of variable factors during the manufacturing process. These factors include the pulverization extent of the medicinal materials, the extraction time, the extraction solvent volume, the alcohol concentration of extraction solvent and extraction temperature. Four marker substances include hesperidin, ferulic acid, cinnamic acid, cinnamaldehyde. The HPLC assay method was used to determine these marker substances in wine. The results showed that the optimal process of producing Ma-Zu-Wan-Show-Yao-Jyo occurred when Citri Reticulatae Pericarpium was pulverized to 0.84 mm in size（# 20 sieve）, Angelicae Sinensis Radix, Chuanxiong Rhizoma and Cinnamomi Cortex was cut to 2.36 mm in size（# 8 sieve）, extracted with 50% alcohol solution, which volume was 10-12 times equivalent to the medicinal materials, then heating at 45 ℃ for 24 hours.

Key words: hesperidin; ferulic acid; cinnamic acid; cinnamaldehyde; Ma-Zu-Wan-Show-Yao-Jyo

Introduction

Chinese tonic wine has been used over thousand years in the history. Up to now, drinking Chinese tonic wine for nourishing our health is widely accepted. However, the prescription to make Chinese tonic wine privately has been passed verbally. No standard formulation is ever made, not mention the validity of the treatment. In this case Department of Health（DOH）,Executive Yuan, R. O. C.（Taiwan）referred to ancient medicine codes and folklore prescriptions and thus regulated twenty-two Chinese tonic wine standard formulations on 2001¹.

The amount of substances extracted from Chinese medicinal materials by alcohol determine the quality of Chinese tonic wine.² DOH on 2001 also announced permission of solvents with different alcohol concentration to produce Chinese tonic wine.¹ The optimal solvent volumes and alcohol concentrations for each standard formulation still require further study. On the other hand, extraction temperature is a recognizable factor affecting release of Chinese medicinal material substances.^2,3 At present, DOH authorizes cold-maceration method and percolation as manufacturing methods. Traditional Chinese medicine codes, however, have prescribed heating methods, such as hot-maceration and decocting method.3 Furthermore, the extraction time is another significant factor. Therefore, the variables during the process of making Chinese tonic wine, such as extraction time, the volume of extraction solvent, the alcohol concentration of extraction solvent and extraction temperature have been discussed by previous studies in Taiwan.^4,5 But the effect on release of active substances by the extent of pulverization for pretreated Chinese medicinal materials, one of the significant factors is still open for discussion.

As a consequence, the study selects a Chinese tonic wine which manufacturing process is needed exploration. And we added a new factor, the pulverization extent for materials to research.

The subject is Chinese tonic wine standard formulation No. 12, Ma-Zu-Wan-Show-Yao-Jyo, which contains 12 Chinese medicinal materials, they are Longan Arillus, Aucklandiae Radix, Amomi Rotundus Fructus, Aquilariae Resinatum Lignum, Gardeniae Fructus, Codonopsis Radix, Angelicae Sinensis Radix, Chuanxiong Rhizoma, Polygonati Odorati Rhizoma, Cinnamomi Cortex, Citri Reticulatae Pericarpium, Acanthopanacis Cortex. Four marker substances include hesperidin（HE）in Citri Reticulatae Pericarpium, ferulic acid（FA）in Angelicae Sinensis Radix and Chuanxiong Rhizoma, cinnamic acid（CA）and cinnamaldehyde（CAD）in Cinnamomi Cortex. The HPLC assay method was used to determine these marker substances in wine, with a view to identifying the effect on release of marker substances and to discover the optimal manufacturing process for Ma-Zu-Wan-Show-Yao-Jyo.^2,7

Materlals and methods

Materials and reagents

The Chinese medicinal materials were all purchased from herbal market. FA was purchased from Acros（New Jersey, USA）. CA was purchased from Nacalai tesque（Kyoto, Japan）.HE, CAD, alcohol acetonitrile were purchased from Merck（Darmstadt, Germany）.

Analytic method

A Hitachi L-7000 system was used. UV wavelength was at 285 nm. Injection volume was 20 μL. The separation was achieved at room temperature using Cosmosil 5C18-AR-Ⅱ column. The mobile phase is shown in Table 1.

Table 1. Gradient elution program using mobile phase A and B

Time （min）	Flow rate （mL/min）	A（%）	B（%）
0	0.8	79	21
12	0.8	79	21
13	1.0	68	32
34	1.0	68	32
35	0.8	79	21

A: water（adjusted to pH 3.0 with phosphoric acid）
B: acetonitrile

Different pulverization extent of pretreated Chinese medicinal materials and extraction time

Take Chinese medicinal materials, one preparation contained Longan Arillus 9.0 g, Aucklandiae Radix 1.0 g, Amomi Rotundus Fructus 2.0 g, Aquilariae Resinatum Lignum 7.0 g, Gardeniae Fructus 3.0 g, Codonopsis Radix 1.0 g, Angelicae Sinensis Radix 1.4 g, Chuanxiong Rhizoma 0.5 g, Polygonati Odorati Rhizoma 3.0 g, Cinnamomi Cortex 1.5 g, Citri Reticulatae Pericarpium 1.0 g, Acanthopanacis Cortex 1.6 g. Cut or pulverize Chinese medicinal materials separately and sieved by a # 8 sieve（2.36 mm）and a # 20 sieve（0.84 mm）. Add 384 mL of 50% alcohol solution and dip under room temperature.

The different volumes of extraction solvent

Take the same material amount of prescription in above paragraph, respectively add 50% alcohol solution 128 mL（4 times）, 192 mL（6 times）, 256 mL（8 times）, 320 mL（10 times）, 384 mL（12 times）, 448 mL（14 times）, 512 mL（16 times）and 576 mL（18 times）under room temperature and dip for 3 weeks.

The different alcohol concentrations of extraction solvent

Take the same material amount of prescription in above paragraph, then respectively measure 10%, 30%, 50%, 60% and 75% alcohol solution as the volume is the optimal one based on the previous test results under room temperature dip for 3 weeks.

The different extraction temperatures

Take the same material amount of prescription in above paragraph, corresponding to the optimal manufacturing condition based on the previous test results. Extract them by dipping for 3 weeks under room temperature, heating for 24 hours at 45℃, heating for 12 hours at 60℃ and boiling（100℃）for 3 hours.

Results and discussion

Different pulverization extent of pretreated Chinese medicinal materials and extraction time

Generally speaking, before preparation, materials require pulverization treatment to increase surface area for dipping. In Table 2, when Citri Reticulatae Pericarpium was pulverized, the released HE was increasing along with duration of extraction time, and reached saturation on the 40th day. By way of cutting for its pretreatment, the saturation reached after 30 days.

Table 2. Content of marker substances in wines which by different pretreatments of Chinese medicinal materials and extraction time.

Days	HE（mg）		FA（mg）		CA（mg）		CAD（mg）
	Cutting	Pulverization	Cutting	Pulverization	Cutting	Pulverization	Cutting	Pulverization
5	8.52 ± 0.22	21.53 ± 1.30	0.90 ± 0.04	0.84 ± 0.02	0.72 ± 0.02	0.55 ± 0.03	9.02 ± 0.11	11.67 ± 0.79
10	12.74 ± 0.18	32.23 ± 0.48	1.17 ± 0.02	1.11 ± 0.02	0.82 ± 0.01	0.59 ± 0.01	10.96 ± 0.14	12.35 ± 0.41
15	14.39 ± 0.56	35.76 ± 0.62	1.33 ± 0.01	1.16 ± 0.01	0.81 ± 0.01	0.59 ± 0.01	11.41 ± 0.23	11.48 ± 0.45
20	18.38 ± 0.27	35.33 ± 0.42	1.47 ± 0.04	1.25 ± 0.01	0.85 ± 0.02	0.59 ± 0.00	13.03 ± 0.37	11.17 ± 0.41
25	19.35 ± 1.56	35.08 ± 0.21	1.31 ± 0.04	1.14 ± 0.03	0.86 ± 0.02	0.62 ± 0.01	13.30 ± 0.39	10.48 ± 0.37
30	22.71 ± 0.30	38.01 ± 0.54	1.37 ± 0.05	1.12 ± 0.01	0.94 ± 0.02	0.61 ± 0.01	15.21 ± 0.28	10.36 ± 0.32
35	23.52 ± 0.64	37.74 ± 0.22	1.19 ± 0.01	1.04 ± 0.00	0.87 ± 0.03	0.64 ± 0.00	14.84 ± 0.49	10.12 ± 0.31
40	28.08 ± 2.02	38.87 ± 0.80	1.23 ± 0.02	1.05 ± 0.02	0.94 ± 0.01	0.61 ± 0.01	16.37 ± 0.16	9.75 ± 0.39
45	26.25 ± 0.46	39.03 ± 0.73	1.22 ± 0.02	1.05 ± 0.02	0.96 ± 0.02	0.64 ± 0.01	16.37 ± 0.38	9.68 ± 0.35
50	26.20 ± 0.64	37.03 ± 0.66	1.31 ± 0.01	1.07 ± 0.03	0.95 ± 0.02	0.60 ± 0.01	16.48 ± 0.43	9.07 ± 0.34
55	27.35 ± 0.58	38.57 ± 0.42	1.34 ± 0.02	1.09 ± 0.02	1.03 ± 0.01	0.69 ± 0.01	18.09 ± 0.26	9.58 ± 0.28
60	29.22 ± 0.71	39.42 ± 0.50	1.37 ± 0.04	1.14 ± 0.01	1.07 ± 0.03	0.67 ± 0.00	18.37 ± 0.19	9.44 ± 0.28

The extracted FA, reached its saturation till the 20th day while the results by means of cutting was better than that of pulverization. FA is sensitive to heat. During pulverization, FA was decomposed by heat, the released quantity was lesser accordingly.⁸

By pulverization, CA released stably after10 days; by cutting, the quantity was growing slowly. In a word, the quantity by means of cutting was more, but longer dipping was necessary. Regarding CAD, the released quantity by way of cutting was growing along with time, but by way of pulverization, attained the maximum quantity after 10 days and decreased slowly later. Finally, compare them after 60- day dipping, the former released 2 times.

So, the extraction of HE preferred pulverization while that of FA, CA and CAD applied cutting. The increase of surface area facilitates substances release from Chinese medicinal materials, yet due to pulverized medicines, cohesion is enforced resulting to inefficient dipping and spreading, adversely hinders release of active substances, even make released substances back.⁷

The different volumes of extraction solvent

All marker substances increased along with the growing volume of solvent, especially for HE（Table 3）. In addition, the released quantity of CAD was saturated when 16 times solvent volume was used and that of FA and CA was saturated when 8 times solvent was used. It is inferred that increasing solvent volume helps extracted quantity of marker substances, but the effect is not in the direct ratio. Moreover, it dilutes unit quantity and increases the cost. Take the cost and the release results of marker substances into consideration, the 10-12 times equivalent to materials is the optimal volume of extracted solvent.

Table 3. Content of marker substances in wines which by different extraction solvent volumes.

Times	HE（mg）	FA（mg）	CA（mg）	CAD（mg）
4	3.96 ± 0.26	1.36 ± 0.01	0.91 ± 0.03	11.91 ± 0.46
6	5.69 ± 0.45	1.59 ± 0.06	1.08 ± 0.06	16.82 ± 0.27
8	6.58 ± 0.55	1.68 ± 0.01	1.41 ± 0.04	20.41 ± 0.29
10	8.13 ± 0.32	1.65 ± 0.02	1.45 ± 0.04	22.24 ± 0.91
12	8.29 ± 0.44	1.67 ± 0.02	1.40 ± 0.02	24.53 ± 0.99
14	9.72 ± 0.59	1.73 ± 0.14	1.41 ± 0.05	28.34 ± 0.40
16	10.97 ± 0.19	1.66 ± 0.11	1.43 ± 0.04	30.74 ± 0.84
18	11.41 ± 0.29	1.78 ± 0.07	1.46 ± 0.05	31.49 ± 0.78

The different alcohol concentrations of extraction solvent

Table 4 indicated FA and CA, which were hydrophilic, released lesser in proportion with growing alcohol. HE and CAD, because of their lipophilic structures, the more alcohol concentration in the solvent, the more extracted quantity. The pH value of the solvent seems also affects some substances. Coniferyl ferulate, one content of Angelicae Sinensis Radix and Chuanxiong Rhizoma, when in neutral and base, strong acid may hydrolysis to be FA⁹. Affected by increasing alcohol concentrations, solvent pH value shifted from weak acid to neutral. It explains why the concentration of alcohol is increasing, the extracted FA is stable instead of lowering. However, based on the outcome of each substance, 50% alcohol shall be the prominent solvent.

Table 4. Content of marker substances in wines which by different alcohol concentrations of solvent.

Alcohol Concentration	pH value	HE（mg）	FA（mg）	CA（mg）	CAD（mg）
10%	5.02	2.77 ± 0.27	3.53 ± 0.19	1.56 ± 0.03	10.60 ± 1.70
15%	5.17	3.55 ± 0.14	2.96 ± 0.05	1.61 ± 0.04	15.74 ± 0.77
30%	5.41	8.50 ± 0.23	1.73 ± 0.12	1.29 ± 0.04	24.06 ± 0.56
50%	5.73	18.85 ± 0.09	1.39 ± 0.02	1.12 ± 0.08	34.22 ± 1.31
60%	5.82	21.02 ± 0.28	1.50 ± 0.05	1.05 ± 0.01	38.32 ± 0.45
75%	5.97	19.38 ± 0.42	1.36 ± 0.08	0.94 ± 0.04	40.41 ± 0.77

The different extraction temperatures

Based on Table 5, after increasing extraction temperature, the concentration of HE is growing affected by soaring temperature. But when the temperature is up, FA is decomposed by heat causing lower concentration.⁸ On the contrary, coniferyl ferulate hydrolysis to be FA leading to increase FA¹⁰. The two results mediate so that the extraction of FA lowers but increases later.

Table 5. Content of marker substances in wins which by different extraction temperatures.

marker substances	Room temperature for 3 weeks （mg）	45℃ for 24 hours （mg）	60℃ for 12 hours （mg）	100℃ for 3hours （mg）
HE	19.36 ± 0.04	30.56 ± 0.02	32.82 ± 0.06	69.83 ± 0.01
FA	2.01 ± 0.09	1.53 ± 0.01	1.43 ± 0.01	1.80 ± 0.00
CA	1.59 ± 0.01	1.06 ± 0.01	0.98 ± 0.01	0.34 ± 0.00
CAD	36.01 ± 0.18	33.66 ± 0.03	31.64 ± 0.04	3.82 ± 0.01

CA decreases along with soaring extraction temperature resulting from decomposition by heat. The concentration of CAD increases by soaring extraction temperature, but when the temperature continues increasing, it drops. Possible reason is that Chinese medicinal materials are heated suddenly, oil secreting cavity is damaged causing volatile oil not dissolving completely in the tonic but evaoporate.¹¹

Increasing temperature to reduce extraction time must concern that active substances may be damaged by heat. Furthermore, after Chinese tonic wine cooling down, extracted substances precipitate due to lower solubility. Meanwhile, plant protein in Chinese medicinal materials condensed and precipitate by heating, causing unclear Chinese tonic wine.7 In our experiment, sediment of the cool wine in sequence of the amount by boiling for 3 hours, 60℃ for 12 hours, 45℃ for 24 hours, room temperature for 3 weeks. With respect to extraction effect and precipitation, heating at 45℃ for 24 hours is the optimal extracting temperature.

Conclusion

In conclusion, the optimal process of producing Ma-Zu-Wan-Show-Yao-Jyo occurred when Citri Reticulatae Pericarpium pulverized to 0.84 mm in size（# 20 sieve）, Angelicae Sinensis Radix, Chuanxiong Rhizoma and Cinnamomi Cortex cut to 2.36 mm in size（# 8 sieve）, extracted with 50% alcohol solution, which volume was 10-12 times relative to the medicinal materials, and then heating at 45 ℃ for 24 hours.

References:

1. Department of Health, Executive Yuan; Notes for unified in tonic wine preparation of chinese herb medicine. Taipei: 2001, No. 090002545.

2. Zhang BQ. Some problems in the production of medicinal liqueur. Liquor-making Science & Technology, 2004; 122: 116-8.

3. Yang JS, Zhang YP, Wu JL, et al: Zhongguo Yaojiu Xue, 1st ed. China, Gui Zhou Science & Technology Press., 2002: pp 102-9.

4. Lay HL, Huang SC, Chen CC, et al: Studies on the component analysis and quality control in tonic wine preparation of King-Mon-Long-Fong-Jyo. J. Food Drugs Anal. 2003; 11: 201-8.

5. Lay HL, Chen CC, Hung SC, et al: Studies on the component and analysis in tonic wine preparation of Shyr-Chyuan-Dah-Buu-Yaw-Jyo. J. Chin. Med. 2006; 1: 47-60.

6. Yanaga A, Goto H, Nakagawa T, et al: Cinnamaldehyde induces endothelium-dependent and -independent vasorelaxant action on isolated rat aorta. Biol. Pharm. Bull. 2006; 29: 2415-8.

7. Zhang LH, Wan HZ. Investigation on the production techniques of health wine. Liquor-making Science & Technolog. 2006; 147: 63-4.

8. Shopora N, Milkova T. Thermal decomposition of α-tetralyl hydroperoxide in the presence of the phenylpropionic acids. Thermochimi. Acta. 1998; 313: 165-174.

9. Lu GH, Chan K, Leung K, et al: Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica Sinensis. J. Chromatogr. A. 2005; 1068: 209-19.

10. Wang YM, Xing ZX, Shi HJ, et al: Determination of ferulic acid in Shenghuatang decoction with different solvent by RP-HPLC. Lishizhen Medicine and Meteria Medica Research. 2007; 18: 2225-6.

11. Zhao HF, Liu SJ, Hou SZ, et al: Study of processing with Cinnamomi Cortex decoction. Shaanxi Journal of Traditional Chinese Medicine. 2007; 28: 731-2.

摘要

在中藥酒的製備過程中，藥材活性成分釋出比例決定中藥酒的品質。在本研究中，以“馬祖”萬壽藥酒為探討對象，了解中藥酒製程中各變因對活性成分釋出之影響情形。這些變因包含了藥材粗細程度、萃取溶媒體積、萃取溶媒酒精含量及萃取溫度等。在不同製程條件下所製備出的中藥酒，我們利用HPLC方法對於製品中4種活性指標成分，包括hesperidin、ferulic acid、cinnamic acid與cinnamaldehyde進行分析。結果顯示，當陳皮的粉碎為0.84 mm（#20篩）；當歸、川芎、肉桂為2.36 mm（#8篩）的情形下，以50%酒精溶液作為萃取溶媒，相對於藥材10-12倍之溶媒體積量，於45℃加熱24小時為其最適化製程。

作者
林雍智藥師
高雄醫學大學藥學系教授 吳秀梅、詹道明